Process of producing an organic compound and an intermediary compound

ABSTRACT

A process of producing an organic compound and/or an intermediary compound by feeding carbon dioxide to a culture of Cyanobacteria cells and subjecting the culture to light, wherein the cells are capable of expressing a nucleic acid molecule that confers the ability to convert a glycolytic intermediate into said organic/intermediary compound. The expression of the nucleic acid molecule is under the control of a regulatory system which responds to a change in the concentration of a nutrient in the culture.

FIELD OF THE INVENTION

The invention relates to a process of producing an organic compoundand/or an intermediary compound produced in the pathway leading to saidorganic compound by feeding carbon dioxide to a culture of acyanobacterial cell and subjecting said culture to light, wherein saidcell is capable of expressing a nucleic acid molecule wherein theexpression of said nucleic acid molecule confers on said cell theability to convert a glycolytic intermediate such as pyruvate orglyceraldehyde 3-phosphate into said organic compound and/or into saidintermediary compound and wherein expression of said nucleic acidmolecule is under the control of a regulatory system which responds to achange in the concentration of a nutrient in said culture. The inventionfurther relates to a cyanobacterial cell for use in this process.

BACKGROUND OF THE INVENTION

Our economy is driven by the use of fossil fuels. Shortages caused byexhausting oil supplies primarily affect the transport sector of oursociety and the chemical industry, but secondarily affect all aspects ofhuman activities. As an additional problem, the use of oil supplies hascaused the build-up of a high CO₂ concentration in the atmosphere.

Energy ultimately comes from the sun and this energy drivesphotosynthetic process in plants and photoautotrophic bacteria. Thisknowledge has led to new methods for the synthesis of biofuels. Inessence, these processes employ plants and algal species to reduce CO₂to the level of sugars and cell material. After harvesting, these endproducts are converted to ethanol by yeast fermentation (in the case ofcrops) or converted chemically to biofuels (in the case of algae). Theoverall energy conservation of these methods is highly inefficient andtherefore demands large surface areas. In addition, the processes arerather labor-intensive, are demanding with respect to water consumptionand affect foodstock prices with adverse consequences for food supplies.A more remotely similar process is based on the conversion of solarenergy into hydrogen. Also this process suffers from a severelydecreased efficiency. Numerous biotechnological processes make use ofgenetically engineered organisms in order to produce bulk or finechemicals, proteins or antibiotics. In many cases, increased productionhas been obtained by improved gene expression and by optimization ofgrowth conditions. In all processes we are aware of, the initialcarbon-precursor has been and still is sugar (notably glucose, but manyother mono- and polysaccharides are in use) or related organicsubstrates: solventogenesis (including butanol and ethanol) and organicacid production (e.g. lactic-, citric- or succinic acid) always startsfrom glucose, which makes it inefficient as the production process usesa high energy initial compound as substrate.

U.S. Pat. No. 6,699,696 describes a process of producing ethanol byfeeding carbon dioxide to a cyanobacterial cell, especially aSynechococcus comprising a nucleic acid molecule encoding an enzymeenabling the cell to convert pyruvate into ethanol, subjecting saidcyanobacterial cell to sun energy and collecting ethanol. This systemhas several drawbacks among others the expression system used istemperature sensitive which demands to adapt the production system forsuch regulation.

Therefore, there is still a need for an alternative and even improvedproduction process of an organic compound, which do not have all thedrawbacks of existing processes.

SUMMARY OF THE INVENTION

The present invention relates to a scalable process for the productionof an organic compound suitable as chemical feedstock or as a biofuel.The invention combines metabolic properties of photoautotrophic andchemoorganotrophic prokaryotes and is based on the employment ofrecombinant oxyphototrophs with high rates of conversion of Calvin cycleintermediates to a fermentative end product. Its novelty resides in thefact a) that its core chemical reactions use CO₂ as the solecarbon-containing precursor and light (preferably sunlight) as the soleenergy source to drive CO₂ reduction and b) that a great variety of endproducts can be realized by the same principle, namely introduction of anucleic acid molecule cassette encoding a specific fermentative pathwayand c) that production is controlled by a medium component and starts atthe most appropriate time, namely at the highest possible cell density.Whereas in current applications of fuel production, organisms aresubstrate (crops in ethanol production) or product (microalgae asbiodiesel), here microorganisms are used as highly specialized catalystsfor the conversion of CO₂ as substrate to a useful end product. Thesecatalysts can be subjected to optimization strategies through physical-and chemical systems-biology approaches. The biochemical background ofthe invention is more extensively described in example 1. Each aspect ofthe invention is more extensively described below.

DETAILED DESCRIPTION OF THE INVENTION Cyanobacteria

In a first aspect, the invention provides a Cyanobacteria capable ofexpressing a nucleic acid molecule, wherein the expression of saidnucleic acid molecule confers on the Cyanobacteria the ability toconvert a glycolytic intermediate into an organic compound and/or intoan intermediary compound produced in the pathway leading to said organiccompound and wherein the nucleic acid molecule is under the control of aregulatory system which responds to a change in the concentration of anutrient when culturing said Cyanobacteria.

In the context of the invention a Cyanobacterium or a cyanobacterialcell is a blue-green algae which is a photosynthetic unicellularprokaryote. Examples of Cyanobacteria include the genera Aphanocapsa,Anabaena, Nostoc, Oscillatoria, Synechococcus, Gloeocapsa, Agmenellum,Scytonema, Mastigocladus, Arthrosprira, Haplosiphon. A preferred genusis Synechococcus. A more preferred species of this genus is aSynechocystis species. Even more preferably, the Synechocystis is aPasteur Culture Collection (PCC) 6083 Synechocystis, which is a publiclyavailable strain via ATCC for example. A preferred organism used is thephototrophic Synechocystis PCC 6083: this is a fast growingcyanobacterium with no specific nutritional demands. Its physiologicaltraits are well-documented: it is able to survive and grow in a widerange of conditions. For example, Synechocystis sp. PCC 6803 can grow inthe absence of photosynthesis if a suitable fixed-carbon source such asglucose is provided. Perhaps most significantly, Synechocystis sp. PCC6803 was the first photosynthetic organism for which the entire genomesequence was determined. In addition, an efficient gene deletionstrategy (Shestakov S V et al, (2002), Photosynthesis Research, 73:279-284 and Nakamura Y et al, (1999), Nucleic Acids Res. 27:66-68) isavailable for Synechocystis sp. PCC 6803, and this organism isfurthermore easily transformable via homologous recombination(Grigirieva G A et al, (1982), FEMS Microbiol. Lett. 13: 367-370).

A Cyanobacteria as defined herein is capable of converting a glycolyticintermediate into an organic compound and/or into an intermediarycompound as defined herein. A biochemical background of theCyanobacteria of the invention is given in Example 1.

A Cyanobacteria as defined herein preferably comprises a nucleic acidmolecule encoding an enzyme capable of converting a glycolyticintermediate into an organic compound and/or into an intermediarycompound as defined herein. An organic compound is herein preferablydefined as being a compound being more reduced than CO₂. A Cyanobacteriais therefore capable of expressing a nucleic acid molecule as definedherein, whereby the expression of a nucleic acid molecule as definedherein confers on the Cyanobacteria the ability to convert a glycolyticintermediate into an organic compound and/or into an intermediarycompound all as defined herein. A glycolytic intermediate may bedihydroxyacetone-phosphate, glyceraldehyde-3-phosphate,1,3-bis-phosphoglycerate, 2-phosphoglycerate, 3-phosphoglycerate,phospho-enol-pyruvate and pyruvate. Preferred glycolytic intermediatesare pyruvate and glyceraldehyde-3-phosphate. The skilled person knowsthat the identity of the glycolytic intermediate converted into anorganic product to be produced depends on the identity of the organicproduct to be produced.

Preferred organic products are selected from: a C1, C2, C3, C4, C5, orC6 alkanol, alkanediol, alkanone, alkene, or organic acid. Preferredalkanols are C2, C3 or C4 alkanols. More preferred are ethanol,propanol, butanol. A preferred alkanediol is 1,3-propanediol. Apreferred alkanone is acetone. A preferred organic acid is D-lactate. Apreferred alkene is ethylene.

A preferred glycolytic intermediate for the production of ethanol,propanol, butanol, acetone or D-lactate is pyruvate. A preferredglycolytic intermediate for the production of 1,3-propanediol isglyceraldehyde-3-phosphate. A preferred glycolytic intermediate for theproduction of ethylene is alpha-oxyglutarate.

“Converting a glycolytic intermediate into an organic compound”preferably means that detectable amounts of an organic compound aredetected in the culture of a Cyanobacteria as defined herein cultured inthe presence of light and dissolved carbon dioxide and/or bicarbonateions during at least 1 day using a suitable assay for the organiccompound. A preferred concentration of said dissolved carbon dioxideand/or bicarbonate ions is at least the natural occurring concentrationat neutral to alkaline conditions (pH 7 to 8) being approximately 1 mM.A more preferred concentration of carbon dioxide and/or bicarbonate ionsis higher than this natural occurring concentration. A preferred methodto increase the carbon dioxide and/or bicarbonate ions in solution is byenrichment with waste carbon dioxide from industrial plants. Theconcentration of carbon dioxide in the gas that is sparged into theculture broth may be increased from the equivalent of 0.03% (air) up to0.2%.

In another preferred embodiment, a Cyanobacterium converts a glycolyticintermediate into an intermediary component of the pathway leading to agiven organic compound. In this embodiment, detectable amounts of anintermediary compound are detected in a Cyanobacterium and/or in itsculture, wherein said Cyanobacterium is cultured in the presence ofsunlight and carbon dioxide during at least 1 day using a given assayfor the intermediary compound. Depending on the identity of the organiccompound, the skilled person will know which intermediary compound maybe produced.

All organic compounds or intermediary compounds produced are producedwithin the cell and may spontaneously diffuse into the culture broth. Apreferred assay for said intermediates and alkanols, alkanones,alkanediols and organic acids is High Performance Liquid Chromatography(HPLC). A detectable amount for said intermediates and alkanols,alkanones, alkanediols and organic acids is preferably at least 0.1 mMunder said culture conditions and using said assay. Preferably, adetectable amount is at least 0.2 mM, 0.3 mM, 0.4 mM, or at least 0.5mM.

Ethanol as Organic Compound

When an organic product to be produced is ethanol, preferred nucleicacid molecules code for enzymes capable of converting pyruvate intoethanol and/or into an intermediary compound produced in the pathwayleading to ethanol, said enzymes comprise a pyruvate decarboxylase (pdc)and an alcohol dehydrogenase (adh). The intermediary compound isacetaldehyde. A preferred assay for acetaldehyde is HPLC. A detectableamount of acetaldehyde is preferably at least 0.1 mM under said cultureconditions as defined earlier herein and using said assay. Therefore inthis preferred embodiment, a Cyanobacterium comprises a nucleic acidmolecule encoding a pdc and another one encoding an adh. Accordingly,this preferred embodiment relates to a Cyanobacterium capable ofexpressing the following nucleic acid molecules being represented bynucleotide sequences, wherein the expression of these nucleotidesequences confers on the cell the ability to convert pyruvate intoacetaldehyde and/or into ethanol:

(a) a nucleotide sequence encoding a pdc, wherein said nucleotidesequence is selected from the group consisting of:

-   -   i. nucleotide sequences encoding a pdc, said pdc comprising an        amino acid sequence that has at least 40% sequence identity with        the amino acid sequence of SEQ ID NO:1.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:2.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code        and        (b) a nucleotide sequence encoding an adh, wherein said        nucleotide sequence is selected from the group consisting of:    -   i. nucleotide sequences encoding an adh, said adh comprising an        amino acid sequence that has at least 40% sequence identity with        the amino acid sequence of SEQ ID NO:3.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:4.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code.

Propanol as Organic Compound

When an organic product to be produced is propanol, the preferrednucleic acid molecules code for enzymes capable of converting pyruvateinto propanol and/or into an intermediary compound produced in thepathway leading to propanol, said enzymes comprise a thiolase, anacetoacetylCoA transferase, an acetoacetate decarboxylase and a propanoldehydrogenase. The intermediary compound is acetone. A preferred assayfor acetone is HPLC. A detectable amount of acetone is preferably atleast 0.1 mM under said culture conditions as defined earlier herein andusing said assay. Therefore in this preferred embodiment, aCyanobacterium comprises a nucleic acid molecule encoding a thiolase, anacetoacetylCoA transferase, an acetoacetate decarboxylase and anotherone encoding a propanol dehydrogenase. Accordingly, this preferredembodiment relates to a Cyanobacterium capable of expressing thefollowing nucleic acid molecules being represented by nucleotidesequences, wherein the expression of these nucleotide sequences conferson the cell the ability to convert pyruvate into acetone and/or intopropanol:

(a) a nucleotide sequence encoding a thiolase, wherein said nucleotidesequence is selected from the group consisting of:

-   -   i. nucleotide sequences encoding a thiolase, said thiolase        comprising an amino acid sequence that has at least 40% sequence        identity with the amino acid sequence of SEQ ID NO:5.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:6.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code,        (b) a nucleotide sequence encoding an acetoacetylCoA        transferase, wherein said nucleotide sequence is selected from        the group consisting of:    -   i. nucleotide sequences encoding an acetoacetylCoA transferase,        said acetoacetylCoA transferase comprising an amino acid        sequence that has at least 40% sequence identity with the amino        acid sequence of SEQ ID NO:95 and another one having the same        sequence identity with SEQ ID NO: 96.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:97 and another one having the same sequence        identity with SEQ ID NO: 98.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code,        (c) a nucleotide sequence encoding an acetoacetylCoA        decarboxylase, wherein said nucleotide sequence is selected from        the group consisting of:    -   i. nucleotide sequences encoding an acetoacetylCoA        decarboxylase, said acetoacetylCoA decarboxylase comprising an        amino acid sequence that has at least 40% sequence identity with        the amino acid sequence of SEQ ID NO:7.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:8.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code        and        (d) a nucleotide sequence encoding a propanol dehydrogenase,        wherein said nucleotide sequence is selected from the group        consisting of:    -   i. nucleotide sequences encoding a propanol dehydrogenase, said        propanol dehydrogenase comprising an amino acid sequence that        has at least 40% sequence identity with the amino acid sequence        of SEQ ID NO:9.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:10.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code.

A preferred acetoacetylCoA transferase is formed by two subunits: one isrepresented by SEQ ID NO:95, the other one by SEQ ID NO:96.Corresponding encoding nucleotide sequences are preferably representedby SEQ ID NO: 97, 98 respectively.

Butanol as Organic Product

Butanol is a preferred organic product. The invention encompasses atleast two pathways for producing butanol.

In a first pathway, when an organic product to be produced is butanol,preferred nucleic acid molecules code for enzymes capable of convertingpyruvate into butanol and/or into an intermediary compound produced inthe pathway leading to butanol, said enzymes comprise a thiolase, ahydroxybutyrylCoA dehydrogenase, a crotonase, a butyryl-CoAdehydrogenase, a butyraldehyde dehydrogenase and a butanoldehydrogenase. A preferred intermediary compound is butyraldehyde. Apreferred assay for butyraldehyde is HPLC. A detectable amount ofbutyraldehyde is at least 0.1 mM under said culture conditions asdefined earlier herein and using said assay. Therefore in this preferredembodiment, a Cyanobacterium comprises a nucleic acid molecule encodinga thiolase, a hydroxybutyrylCoA dehydrogenase, a crotonase, abutyryl-CoA dehydrogenase, a butyraldehyde dehydrogenase and a butanoldehydrogenase. Accordingly, this preferred embodiment relates to aCyanobacterium capable of expressing the following nucleotide moleculesbeing represented by nucleotide sequences, wherein the expression ofthese nucleotide sequences confers on the cell the ability to convertpyruvate into butyraldehyde and/or into butanol:

(a) a nucleotide sequence encoding a thiolase, wherein said nucleotidesequence is selected from the group consisting of:

-   -   i. nucleotide sequences encoding a thiolase, said thiolase        comprising an amino acid sequence that has at least 40% sequence        identity with the amino acid sequence of SEQ ID NO:11.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:12.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code,        (b) a nucleotide sequence encoding a hydroxybutyrylCoA        dehydrogenase, wherein said nucleotide sequence is selected from        the group consisting of:    -   i. nucleotide sequences encoding an hydroxybutyrylCoA        dehydrogenase, said hydroxybutyrylCoA dehydrogenase comprising        an amino acid sequence that has at least 40% sequence identity        with the amino acid sequence of SEQ ID NO:13.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:14.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code,        (c) a nucleotide sequence encoding a crotonase, wherein said        nucleotide sequence is selected from the group consisting of:    -   i. nucleotide sequences encoding a crotonase, said crotonase        comprising an amino acid sequence that has at least 40% sequence        identity with the amino acid sequence of SEQ ID NO:15.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:16.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code,        (d) a nucleotide sequence encoding a butyryl-CoA dehydrogenase,        wherein said nucleotide sequence is selected from the group        consisting of:    -   i. nucleotide sequences encoding a butyryl-CoA dehydrogenase,        said butyryl-CoA dehydrogenase comprising an amino acid sequence        that has at least 40% sequence identity with the amino acid        sequence of SEQ ID NO:17.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:18.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code,        (e) a nucleotide sequence encoding an butyraldehyde        dehydrogenase, wherein said nucleotide sequence is selected from        the group consisting of:    -   i. nucleotide sequences encoding a butyraldehyde dehydrogenase,        said butyraldehyde dehydrogenase comprising an amino acid        sequence that has at least 40% sequence identity with the amino        acid sequence of SEQ ID NO:19.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:20.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code        and        (f) a nucleotide sequence encoding an butanol dehydrogenase,        wherein said nucleotide sequence is selected from the group        consisting of:    -   i. nucleotide sequences encoding a butanol dehydrogenase, said        butanol dehydrogenase comprising an amino acid sequence that has        at least 40% sequence identity with the amino acid sequence of        SEQ ID NO:21.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:22.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code.

In a second pathway, when an organic product to be produced is butanol,preferred nucleic acid molecules code for enzymes capable of convertingpyruvate into butanol and/or into an intermediary compound produced inthe pathway leading to butanol, said enzymes comprise a 2-acetolactatesynthetase, an acetolactate decarboxylase, a diacetyl reductase, anacetoin reductase, a glycerol dehydratase (a large, medium and smallsubunits thereof), a 1,3-propanediol dehydrogenase.

A preferred intermediary compound is 2,3-butanediol. A preferred assayfor 2,3-butanediol is HPLC. A detectable amount of 2,3-butanediol is atleast 0.1 mM under said culture conditions as defined earlier herein andusing said assay. Therefore in this preferred embodiment, aCyanobacterium comprises a nucleic acid molecule encoding a2-acetolactate synthetase, an acetolactate decarboxylase, a diacetylreductase, an acetoin reductase, a glycerol dehydratase (a large, mediumand small subunits thereof), a 1,3-propanediol dehydrogenase.

Accordingly, this preferred embodiment relates to a Cyanobacteriumcapable of expressing the following nucleotide molecules beingrepresented by nucleotide sequences, wherein the expression of thesenucleotide sequences confers on the cell the ability to convert pyruvateinto 2,3-butanediol and/or into butanol:

(a) a nucleotide sequence encoding a 2-acetolactate synthetase, whereinsaid nucleotide sequence is selected from the group consisting of:

-   -   i. nucleotide sequences encoding a 2-acetolactate synthetase,        said 2-acetolactate synthetase e comprising an amino acid        sequence that has at least 40% sequence identity with the amino        acid sequence of SEQ ID NO:75.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:76.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code,        (b) a nucleotide sequence encoding an acetolactate        decarboxylase, wherein said nucleotide sequence is selected from        the group consisting of:    -   i. nucleotide sequences encoding an acetolactate decarboxylase,        said acetolactate decarboxylase comprising an amino acid        sequence that has at least 40% sequence identity with the amino        acid sequence of SEQ ID NO:77.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:78.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code,        (c) a nucleotide sequence encoding a diacetyl reductase, wherein        said nucleotide sequence is selected from the group consisting        of:    -   i. nucleotide sequences encoding a diacetyl reductase, said        diacetyl reductase comprising an amino acid sequence that has at        least 40% sequence identity with the amino acid sequence of SEQ        ID NO:79    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:80    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code,        (d) a nucleotide sequence encoding an acetoin reductase, wherein        said nucleotide sequence is selected from the group consisting        of:    -   i. nucleotide sequences encoding an acetoin reductase, said        acetoin reductase comprising an amino acid sequence that has at        least 40% sequence identity with the amino acid sequence of SEQ        ID NO:81.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:82.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code,        (e) a nucleotide sequence encoding a glycerol dehydratase (i.e.        a large, medium and small subunit thereof), wherein said        nucleotide sequence is selected from the group consisting of:    -   i. nucleotide sequences encoding a large, medium and small        subunits of a glycerol dehydratase, said large, medium and small        subunits of a glycerol dehydratase comprising an amino acid        sequence that has at least 40% sequence identity with the amino        acid sequence of SEQ ID NO:83, 84 and 85 respectively.    -   ii. nucleotide sequences encoding a large, medium and small        subunits of said enzyme, said nucleotide sequence comprising a        nucleotide sequence that has at least 40% sequence identity with        the nucleotide sequence of SEQ ID NO:86, 87 and 88 respectively.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code        and        (f) a nucleotide sequence encoding a 1,3-propanediol        dehydrogenase, wherein said nucleotide sequence is selected from        the group consisting of:    -   i. nucleotide sequences encoding a 1,3-propanediol        dehydrogenase, said 1,3-propanediol dehydrogenase. comprising an        amino acid sequence that has at least 40% sequence identity with        the amino acid sequence of SEQ ID NO:89.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:90.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code.

Acetone as Organic Product

When an organic product to be produced is acetone, preferred nucleicacid molecules code for enzymes capable of converting pyruvate intoacetone and/or into an intermediary compound produced in the pathwayleading to acetone, said enzymes comprise a thiolase, an acetoacetylCoAtransferase and an acetoacetate decarboxylase. Therefore, in thispreferred embodiment, a Cyanobacterium comprises a nucleic acid moleculeencoding a thiolase, an acetoacetylCoA transferase and another oneencoding an acetoacetylCoA decarboxylase. Accordingly, this preferredembodiment relates to a Cyanobacterium capable of expressing thefollowing nucleic acid molecules being represented by nucleotidesequences, wherein the expression of these nucleotide sequences conferson the cell the ability to convert pyruvate into acetone:

(a) a nucleotide sequence encoding a thiolase, wherein said nucleotidesequence is selected from the group consisting of:

-   -   i. nucleotide sequences encoding a thiolase, said thiolase        comprising an amino acid sequence that has at least 40% sequence        identity with the amino acid sequence of SEQ ID NO:23.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:24.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code,        (b) a nucleotide sequence encoding an acetoacetylCoA        transferase, wherein said nucleotide sequence is selected from        the group consisting of:    -   i. nucleotide sequences encoding an acetoacetylCoA transferase,        said acetoacetylCoA transferase comprising an amino acid        sequence that has at least 40% sequence identity with the amino        acid sequence of SEQ ID NO:95 and another one having the same        sequence identity with SEQ ID NO: 96.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:97 and another one having the same sequence        identity with SEQ ID NO: 98.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code and        (c) a nucleotide sequence encoding an acetoacetylCoA        decarboxylase, wherein said nucleotide sequence is selected from        the group consisting of:    -   i. nucleotide sequences encoding an acetoacetylCoA        decarboxylase, said acetoacetylCoA decarboxylase comprising an        amino acid sequence that has at least 40% sequence identity with        the amino acid sequence of SEQ ID NO:25.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:26.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code.

1,3-Propanediol as Organic Product

When an organic product to be produced is 1,3-propanediol, preferrednucleic acid molecules code for enzymes capable of convertingglyceraldehyde-3-phosphate into propanediol and/or into an intermediarycompound produced in the pathway leading to 1,3-propanediol, saidenzymes comprise a glycerol-3-P-dehydrogenase, aglycerol-3-P-phosphatase, a glycerol dehydratase and an oxidoreductase.A first intermediary product is glycerol. A preferred assay for glycerolis HPLC. A detectable amount of glycerol is preferably at least 0.1 mMunder said culture conditions as defined earlier herein and using saidassay. A second intermediary product is hydroxypronionaldehyde. Apreferred assay for hydroxypropionaldehyde is HPLC. A detectable amountof hydroxypropionaldehyde is preferably at least 0.1 mM under saidculture conditions as defined earlier herein and using said assay.Alternatively, a cell may produce a combination of a first and a secondintermediary product as defined above. In this case, a detectable amountof a first and a second intermediary product as defined above is atleast 0.1 mM of each intermediary product. Therefore in this preferredembodiment, a

Cyanobacterium comprises a nucleic acid molecule encoding aglycerol-3-P-dehydrogenase, a glycerol-3-P-phosphatase, a glyceroldehydratase and an oxidoreductase. Accordingly, this preferredembodiment relates to a Cyanobacterium capable of expressing thefollowing nucleic acid molecules being represented by nucleotidesequences, wherein the expression of these nucleotide sequences conferson the cell the ability to convert glyceraldehyde-3-phosphate intoglycerol, hydroxypropionaldehyde and/or 1,3-propanediol:

(a) a nucleotide sequence encoding a glycerol-3-P-dehydrogenase, whereinsaid nucleotide sequence is selected from the group consisting of:

-   -   i. nucleotide sequences encoding a glycerol-3-P-dehydrogenase,        said glycerol-3-P-dehydrogenase comprising an amino acid        sequence that has at least 40% sequence identity with the amino        acid sequence of SEQ ID NO:27.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:28.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code,        (b) a nucleotide sequence encoding a glycerol-3-P-phosphatase,        wherein said nucleotide sequence is selected from the group        consisting of:    -   i. nucleotide sequences encoding a glycerol-3-P-phosphatase,        said glycerol-3-P-phosphatase comprising an amino acid sequence        that has at least 40% sequence identity with the amino acid        sequence of SEQ ID NO:29.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:30.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code,        (c) a nucleotide sequence encoding a glycerol dehydratase,        wherein said nucleotide sequence is selected from the group        consisting of:    -   i. nucleotide sequences encoding a glycerol dehydratase, said        glycerol dehydratase comprising an amino acid sequence that has        at least 40% sequence identity with the amino acid sequence of        SEQ ID NO:31.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:32.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code,        and        (d) a nucleotide sequence encoding an oxidoreductase, wherein        said nucleotide sequence is selected from the group consisting        of:    -   i. nucleotide sequences encoding an oxidoreductase, said        oxidoreductase comprising an amino acid sequence that has at        least 40% sequence identity with the amino acid sequence of SEQ        ID NO:33.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:34.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code.

D-Lactate as Organic Product

When an organic product to be produced is D-lactate, preferred nucleicacid molecules code for enzymes capable of converting pyruvate intoD-lactate, said enzyme comprise a lactate dehydrogenase. Preferredassays for D-lactate are HPLC and enzymatic assays. A detectable amountby HPLC of D-lactate is preferably at least 0.1 mM under said cultureconditions as defined earlier herein and using said assay. A detectableamount by enzymatic assays of D-lactate is preferably at least 0.2 mg/lunder said culture conditions as defined earlier herein and using saidassay. Therefore, in this preferred embodiment, a Cyanobacteriumcomprises a nucleic acid molecule encoding a lactate dehydrogenase.Accordingly, this preferred embodiment relates to a Cyanobacteriumcapable of expressing at least one nucleic acid molecule, said nucleicacid molecule being represented by a nucleotide sequence, wherein theexpression of this nucleotide sequence confers on the cell the abilityto convert pyruvate into D-lactate:

(a) a nucleotide sequence encoding a lactate dehydrogenase, wherein saidnucleotide sequence is selected from the group consisting of:

-   -   i. nucleotide sequences encoding a lactate dehydrogenase, said        lactate dehydrogenase comprising an amino acid sequence that has        at least 40% sequence identity with the amino acid sequence of        SEQ ID NO:35.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:36.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code.

Ethylene as Organic Product

When an organic product to be produced is ethylene, preferred nucleicacid molecules code for enzymes capable of converting 2-oxoglutarateinto ethylene and succinate, said enzyme comprise a ethylene formingenzyme (2-oxoglutarate-dependent ethylene-forming enzyme). A preferredassay for ethylene is GC (gas chromatography) under said cultureconditions as defined earlier herein and using said assay. As shown by(Pirkov I et al, (2008), Metabolic Engineering, 10: 276-280). Adetectable amount of ethylene is preferably at least 10 μg/1 h.Therefore, in this preferred embodiment, a Cyanobacterium comprises anucleic acid molecule encoding a ethylene forming enzyme. Accordingly,this preferred embodiment relates to a Cyanobacterium capable ofexpressing at least one nucleic acid molecule, said nucleic acidmolecule being represented by a nucleotide sequence, wherein theexpression of this nucleotide sequence confers on the cell the abilityto convert 2-oxoglutarate into ethylene and succinate:

(a) a nucleotide sequence encoding a ethylene forming enzyme, whereinsaid nucleotide sequence is selected from the group consisting of:

-   -   i. nucleotide sequences encoding a ethylene forming enzyme, said        ethylene forming enzyme comprising an amino acid sequence that        has at least 40% sequence identity with the amino acid sequence        of SEQ ID NO:91.    -   ii. nucleotide sequences comprising a nucleotide sequence that        has at least 40% sequence identity with the nucleotide sequence        of SEQ ID NO:92.    -   iii. nucleotide sequences the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii);    -   iv. nucleotide sequences the sequences of which differs from the        sequence of a nucleic acid molecule of (iii) due to the        degeneracy of the genetic code.

Each nucleotide sequence or amino acid sequence described herein byvirtue of its identity percentage (at least 40%) with a given nucleotidesequence or amino acid sequence respectively has in a further preferredembodiment an identity of at least 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98%, 99% or more identity with the given nucleotideor amino acid sequence respectively. In a preferred embodiment, sequenceidentity is determined by comparing the whole length of the sequences asidentified herein.

Each nucleotide sequence encoding an enzyme as described herein mayencode either a prokaryotic or an eukaryotic enzyme, i.e. an enzyme withan amino acid sequence that is identical to that of an enzyme thatnaturally occurs in a prokaryotic or eukaryotic organism. The presentinventors have found that the ability of a particular enzyme or to acombination of particular enzymes as defined herein to confer to aCyanobacterial cell the ability to convert a glycolytic intermediateinto an organic product and/or into an intermediary compound produced inthe pathway leading to the organic compound does not depend so much onwhether the enzyme is of prokaryotic or eukaryotic origin. Rather thisdepends on the relatedness (identity percentage) of the enzyme aminoacid sequence or corresponding nucleotide sequence to that of thecorresponding identified SEQ ID NO.

Alternatively or in combination with previous preferred embodiments, theinvention relates to a further preferred embodiment, wherein at leastone enzyme as defined herein is substantially not sensitive towardsoxygen inactivation. “Being substantially not sensitive towards oxygeninactivation” preferably means that when such enzyme is expressed in aCyanobacterium as described herein and when this Cyanobacterium iscultured in a process of the invention, significant activity of saidenzyme is detectable using a specific assay known to the skilled person.More preferably, a significant activity of said enzyme is at least 20%of the activity of the same enzyme expressed in the same Cyanobacteriumbut cultured in the absence of oxygen. Even more preferably, at least30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% ofthe activity is detectable. Most preferably, the activity of said enzymeas expressed in a Cyanobacterium as described herein and when thisCyanobacterium is cultured in the process of the invention is identicalwith the activity of the same enzyme as expressed in a sameCyanobacterium as described herein and when this Cyanobacterium iscultured in the absence of oxygen. This is an advantage of the presentinvention that the Cyanobacterium hence obtained is preferably used in aprocess of the invention wherein oxygen is produced, since it willsubstantially not affect the activity of the enzymes used herein.

Alternatively or in combination with previous preferred embodiments, theinvention relates to a further preferred embodiment wherein, aCyanobacterium as defined herein is a Cyanobacterium that has beentransformed with a nucleic acid construct comprising a nucleotidesequence encoding an enzyme as defined above depending on the organicproduct to be produced. A nucleic acid construct comprising a nucleicacid molecule coding for a given enzyme as defined herein will ensureexpression of the given nucleic acid molecule, and of the correspondingenzyme in a Cyanobacterium. In a more preferred embodiment, a nucleicacid construct comprises more than one nucleic acid molecule, eachnucleic acid molecule coding for a given enzyme. In an even morepreferred embodiment, a nucleic acid construct comprises two, three,four nucleic acid molecules, each nucleic acid molecule coding for agiven enzyme. In a most preferred embodiment, a nucleic acid constructcomprises all nucleic acid molecules needed for the conversion of aglycolytic intermediate into an organic product and/or an intermediarycompound, each nucleic acid molecule coding for a given enzyme. Thismost preferred embodiment is illustrated in example 2. In this mostpreferred embodiment, a nucleic acid construct comprises an expressioncassette, said expression cassette comprising each needed nucleic acidmolecule. Each nucleic acid molecule is operably linked with othernucleic acid molecule present. Most preferably, a suitable promoter isoperably linked with the expression cassette to ensure expression of thenucleic acid molecule in a Cyanobacterium as later defined herein.

To this end, a nucleic acid construct may be constructed as described ine.g. U.S. Pat. No. 6,699,696 or 4,778,759. A Cyanobacterium may comprisea single but preferably comprises multiple copies of each nucleic acidconstruct. A nucleic acid construct may be maintained episomally andthus comprises a sequence for autonomous replication, such as an ARSsequence. Suitable episomal nucleic acid constructs may e.g. be based onthe yeast 2μ or pKD1 (Fleer et al., 1991, Biotechnology 9:968-975)plasmids. Preferably, however, each nucleic acid construct is integratedin one or more copies into the genome of a cyanobacterial cell.Integration into a cyanobacterial cell's genome may occur at random byillegitimate recombination but preferably a nucleic acid construct isintegrated into the Cyanobacterium cell's genome by homologousrecombination as is well known in the art (U.S. Pat. No. 4,778,759).Homologous recombination occurs preferably at a neutral integrationsite. A neutral integration site is an integration which is not expectedto be necessary for the production process of the invention, i.e for thegrowth and/or the production of an organic compound and/or anintermediary compound as defined herein. A preferred integration site isthe nrt operon as illustrated in the examples (Osanai, T., Imamura, S.,Asayama, M., Shirai, M., Suzuki, I., Murata, N., Tanaka, K, (2006)Nitrogen induction of sugar catabolic gene expression in Synechocystissp. PCC 6803. DNA Research 13, 185-19).

Accordingly, in a more preferred embodiment, a cyanobacterial cell ofthe invention comprises a nucleic acid construct comprising a nucleicacid molecule, said nucleic acid molecule being represented by anucleotide sequence, said nucleotide sequence being a coding sequence ofan enzyme as identified herein. Said cyanobacterial cell is capable ofexpression of these enzymes. In an even more preferred embodiment, anucleic acid molecule encoding an enzyme is operably linked to apromoter that causes sufficient expression of a corresponding nucleicacid molecule in a Cyanobacterium to confer to a Cyanobacterium theability to convert a glycolytic intermediate into a given organicproduct and/or into an intermediary compound produced in the pathwayleading to the organic product. In case of an expression cassette asearlier defined herein, a promoter is upstream of the expressioncassette. Accordingly, in a further aspect, the invention alsoencompasses a nucleic acid construct as earlier outlined herein.Preferably, a nucleic acid construct comprises a nucleic acid moleculeencoding an enzyme as earlier defined herein. Nucleic acid moleculesencoding an enzyme have been all earlier defined herein.

A promoter that could be used to achieve the expression of a nucleicacid molecule coding for an enzyme as defined herein may be not nativeto a nucleic acid molecule coding for an enzyme to be expressed, i.e. apromoter that is heterologous to the nucleic acid molecule (codingsequence) to which it is operably linked. Although a promoter preferablyis heterologous to a coding sequence to which it is operably linked, itis also preferred that a promoter is homologous, i.e. endogenous to aCyanobacterium. Preferably, a heterologous promoter (to the nucleotidesequence) is capable of producing a higher steady state level of atranscript comprising a coding sequence (or is capable of producing moretranscript molecules, i.e. mRNA molecules, per unit of time) than is apromoter that is native to a coding sequence, preferably underconditions where sun light and carbon dioxide are present. A suitablepromoter in this context includes both constitutive and induciblenatural promoters as well as engineered promoters. A promoter used in aCyanobacterium cell of the invention may be modified, if desired, toaffect its control characteristics. A preferred promoter is a SigEcontrolled promotor of the glyceraldehyde dehydrogenase gene fromSynechocystis PCC 6083 as identified in SEQ ID NO:74 (Takashi Osanai, etal, Positive Regulation of Sugar Catabolic Pathways in theCyanobacterium Synechocystis sp. PCC 6803 by the Group 2 sigma FactorSigE. J. Biol. Chem. (2005) 35: 30653-30659). This promoter is quiteadvantageous to be used as outlined below in the next paragraph.

Alternatively or in combination with previous preferred embodiments, theinvention relates to a further preferred embodiment, wherein theexpression of a nucleic acid molecule as defined herein is regulated soas to respond to a change in the concentration of a nutrient such asammonium (Osanai, T., Imamura, S., Asayama, M., Shirai, M., Suzuki, I.,Murata, N., Tanaka, K, (2006) Nitrogen induction of sugar catabolic geneexpression in Synechocystis sp. PCC 6803. DNA Research 13, 185-195).

In a more preferred embodiment, the expression of a nucleic acidmolecule is induced when ammonium concentration is below a given value.As exemplified in example 2, this is preferably achieved by using a SigEpromoter in a nucleic acid construct comprising a nucleic acid moleculeas defined herein. Such promoter is inactive in a first phase of theprocess when ammonium is present in a concentration which isapproximately above 1 mM. In this first phase, a Cyanobacterium willgrow and not produce any organic compound and/or any intermediarycompound as defined herein. When the ammonium source, has been used forgrowth and its concentration is approximately below 1 mM, the SigEpromoter is induced. As a consequence, the process is divided in 2phase, a first phase where cell numbers increase and a second phase ofthe production process of the invention, which is characterized by theproduction of an organic compound and/or an intermediary compound asdefined herein. This two phased production process has severaladvantages compared to one phase production processes: a) the growthphase is separated from the production phase and therefore high celldensities can be obtained in a short time b) the yield of an organicproduct and/or of an intermediary compound as defined herein will beimproved due to the fact that no carbon flux to growth will occur in thesecond phase. The skilled person knows how to assess the concentrationof a nutrient such as ammonium in the culture.

Method

In a second aspect, the invention relates to a process of producing anorganic compound and/or an intermediary compound as defined herein byfeeding carbon dioxide to a culture of a cyanobacterial cell andsubjecting said culture to light, wherein said cell is capable ofexpressing a nucleic acid molecule, wherein the expression of saidnucleic acid molecule confer on the cell the ability to convert aglycolytic intermediate into an organic compound and/or into anintermediary compound produced in the pathway leading to the organiccompound and wherein said nucleic acid molecule is under the control ofa regulatory system which responds to a change in the concentration of anutrient in said culture. A Cyanobacterium, a glycolytic intermediate,an organic compound, an intermediary compound, a nucleic acid molecule,and a regulatory system have all earlier been defined herein.

In a process of the invention, carbon dioxide is fed to a culture brothof Cyanobacteria. The skilled person knows that the carbon dioxideconcentration is dependent from the temperature, the pH and theconcentration of carbon dioxide present in the air used. Therefore, thisis quite difficult to give an estimation of the concentration of carbondioxide which is being used. Below, we give estimations of preferredconcentrations used. A preferred feeding concentration of carbon dioxideis air enriched to 5% carbon dioxide. A preferred source of carbondioxide may be the waste gas from an industrial plant.

Usually a process is started with a culture (also named culture broth)of Cyanobacteria having an optical density measured at 660 nm ofapproximately 0.2 to 2.0 (OD₆₆₀=0.2 to 2) as measured in anyconventional spectrophotometer with a measuring path length of 1 cm.Usually the cell number in the culture doubles every 20 hours. Apreferred process takes place in a tank with a depth of 30-50 cm exposedto sun light. In a preferred process, the number of cells increasesuntil the source of ammonium is exhausted or below a given value asearlier explained herein, subsequently the production of said productsand/or intermediates will start. In a preferred embodiment, the lightused is natural. A preferred natural light is sunlight. Sunlight mayhave an intensity ranged between approximately 1000 and approximately1500 μEinstein/m²/s. In another preferred embodiment, the light used isartificial. Such artificial light may have an intensity ranged betweenapproximately 70 and approximately 800 μEinstein/m²/s.

In a preferred process, an organic compound and/or an intermediatecompound produced is separated from the culture broth. This may berealized continuously with the production process or subsequently to it.Separation may be based on membrane technology and/or evaporationmethods. Depending on the identity of the organic compound and/or ofintermediary compound produced, the skilled person will know whichseparating method is the most appropriate.

General Definitions Sequence Identity and Similarity

Sequence identity is herein defined as a relationship between two ormore amino acid (polypeptide or protein) sequences or two or morenucleic acid (polynucleotide) sequences, as determined by comparing thesequences. Usually, sequence identities or similarities are comparedover the whole length of the sequences compared. In the art, “identity”also means the degree of sequence relatedness between amino acid ornucleic acid sequences, as the case may be, as determined by the matchbetween strings of such sequences. “Similarity” between two amino acidsequences is determined by comparing the amino acid sequence and itsconserved amino acid substitutes of one polypeptide to the sequence of asecond polypeptide. “Identity” and “similarity” can be readilycalculated by various methods, known to those skilled in the art. In apreferred embodiment, sequence identity is determined by comparing thewhole length of the sequences as identified herein.

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include e.g. the BestFit, BLASTP (Protein BasicLocal Alignment Search Tool), BLASTN (Nucleotide Basic Local AlignmentSearch Tool), and FASTA (Altschul, S. F. et al., J. Mol. Biol.215:403-410 (1990), publicly available from NCBI and other sources(BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894).A most preferred algorithm used is EMBOSS (European Molecular BiologyOpen Software Suite). Preferred parameters for amino acid sequencescomparison using EMBOSS are gap open 10.0, gap extend 0.5, Blosum 62matrix. Preferred parameters for nucleic acid sequences comparison usingEMBOSS are gap open 10.0, gap extend 0.5, DNA full matrix (DNA identitymatrix).

Optionally, in determining the degree of amino acid similarity, theskilled person may also take into account so-called “conservative” aminoacid substitutions, as will be clear to the skilled person. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulphur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. Substitutional variants of the amino acid sequencedisclosed herein are those in which at least one residue in thedisclosed sequences has been removed and a different residue inserted inits place. Preferably, the amino acid change is conservative. Preferredconservative substitutions for each of the naturally occurring aminoacids are as follows: Ala to ser; Arg to lys; Asn to gln or his; Asp toglu; Cys to ser or ala; Gln to asn; Glu to asp; Gly to pro; His to asnor gln; Ile to leu or val; Leu to ile or val; Lys to arg; gln or glu;Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trpto tyr; Tyr to trp or phe; and, Val to ile or leu.

Hybridising Nucleic Acid Sequences

Nucleotide sequences encoding the enzymes expressed in the cell of theinvention may also be defined by their capability to hybridise with thenucleotide sequences of SEQ ID NO: 2, 4, 6, 8, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 76, 78, 80, 82, 86, 87, 88, 90, 92, 96 respectively,under moderate, or preferably under stringent hybridisation conditions.Stringent hybridisation conditions are herein defined as conditions thatallow a nucleic acid sequence of at least about 25, preferably about 50nucleotides, 75 or 100 and most preferably of about 200 or morenucleotides, to hybridise at a temperature of about 65° C. in a solutioncomprising about 1 M salt, preferably 6×SSC or any other solution havinga comparable ionic strength, and washing at 65° C. in a solutioncomprising about 0.1 M salt, or less, preferably 0.2×SSC or any othersolution having a comparable ionic strength. Preferably, thehybridisation is performed overnight, i.e. at least for 10 hours andpreferably washing is performed for at least one hour with at least twochanges of the washing solution. These conditions will usually allow thespecific hybridisation of sequences having about 90% or more sequenceidentity. Moderate conditions are herein defined as conditions thatallow a nucleic acid sequences of at least 50 nucleotides, preferably ofabout 200 or more nucleotides, to hybridise at a temperature of about45° C. in a solution comprising about 1 M salt, preferably 6×SSC or anyother solution having a comparable ionic strength, and washing at roomtemperature in a solution comprising about 1 M salt, preferably 6×SSC orany other solution having a comparable ionic strength. Preferably, thehybridisation is performed overnight, i.e. at least for 10 hours, andpreferably washing is performed for at least one hour with at least twochanges of the washing solution. These conditions will usually allow thespecific hybridisation of sequences having up to 50% sequence identity.The person skilled in the art will be able to modify these hybridisationconditions in order to specifically identify sequences varying inidentity between 50% and 90%.

Homologous

The term “homologous” when used to indicate the relation between a given(recombinant) nucleic acid or polypeptide molecule and a given hostorganism or host cell, is understood to mean that in nature the nucleicacid or polypeptide molecule is produced by a host cell or organisms ofthe same species, preferably of the same variety or strain. Ifhomologous to a host cell, a nucleic acid sequence encoding apolypeptide will typically be operably linked to another promotersequence than in its natural environment. When used to indicate therelatedness of two nucleic acid sequences the term “homologous” meansthat one single-stranded nucleic acid sequence may hybridize to acomplementary single-stranded nucleic acid sequence. The degree ofhybridization may depend on a number of factors including the amount ofidentity between the sequences and the hybridization conditions such astemperature and salt concentration as earlier presented. Preferably theregion of identity is greater than about 5 bp, more preferably theregion of identity is greater than 10 bp. Preferably, two nucleic acidor polypeptides sequences are said to be homologous when they have morethan 80% identity.

Heterologous

The term “heterologous” when used with respect to a nucleic acid (DNA orRNA) or protein refers to a nucleic acid or protein (also namedpolypeptide or enzyme) that does not occur naturally as part of theorganism, cell, genome or DNA or RNA sequence in which it is present, orthat is found in a cell or location or locations in the genome or DNA orRNA sequence that differ from that in which it is found in nature.Heterologous nucleic acids or proteins are not endogenous to the cellinto which it is introduced, but has been obtained from another cell orsynthetically or recombinantly produced. Generally, though notnecessarily, such nucleic acids encode proteins that are not normallyproduced by the cell in which the DNA is transcribed or expressed.Similarly exogenous RNA encodes for proteins not normally expressed inthe cell in which the exogenous RNA is present. Heterologous nucleicacids and proteins may also be referred to as foreign nucleic acids orproteins. Any nucleic acid or protein that one of skill in the art wouldrecognize as heterologous or foreign to the cell in which it isexpressed is herein encompassed by the term heterologous nucleic acid orprotein. The term heterologous also applies to non-natural combinationsof nucleic acid or amino acid sequences, i.e. combinations where atleast two of the combined sequences are foreign with respect to eachother.

Operably Linked

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements (or coding sequences or nucleic acid sequence ornucleic acid molecule) in a functional relationship. A nucleic acidsequence is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the coding sequence. Operably linked meansthat the nucleic acid sequences being linked are typically contiguousand, where necessary to join two protein coding regions, contiguous andin reading frame.

Promoter

As used herein, the term “promoter” refers to a nucleic acid fragmentthat functions to control the transcription of one or more nucleic acidmolecules, located upstream with respect to the direction oftranscription of the transcription initiation site of the nucleic acidmolecule, and is structurally identified by the presence of a bindingsite for DNA-dependent RNA polymerase, transcription initiation sitesand any other DNA sequences, including, but not limited to transcriptionfactor binding sites, repressor and activator protein binding sites, andany other sequences of nucleotides known to one of skill in the art toact directly or indirectly to regulate the amount of transcription fromthe promoter. A “constitutive” promoter is a promoter that is activeunder most environmental and developmental conditions. An “inducible”promoter is a promoter that is active under environmental ordevelopmental regulation.

Genetic Modifications

For overexpression of an enzyme in a host cells=of the inventions asdescribed above, as well as for additional genetic modification of ahost cell=, preferably Cyanobacteria, host cells are transformed withthe various nucleic acid constructs of the invention by methods wellknown in the art. Such methods are e.g. known from standard handbooks,such as Sambrook and Russel (2001) “Molecular Cloning: A LaboratoryManual (3^(rd) edition), Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, or F. Ausubel et al, eds., “Current protocolsin molecular biology”, Green Publishing and Wiley Interscience, New York(1987). Methods for transformation and genetic modification ofcyanobacterial cells are known from e.g. U.S. Pat. No. 6,699,696 or4,778,759.

A promoter for use in a nucleic acid construct for overexpression of anenzyme in a cyanobacterial cell of the invention has been describedabove. Optionally, a selectable marker may be present in a nucleic acidconstruct. As used herein, the term “marker” refers to a gene encoding atrait or a phenotype which permits the selection of, or the screeningfor, a Cyanobacterial cell containing the marker. A marker gene may bean antibiotic resistance gene whereby the appropriate antibiotic can beused to select for transformed cells from among cells that are nottransformed. Preferably however, a non-antibiotic resistance marker isused, such as an auxotrophic marker (URA3, TRP1, LEU2). In a preferredembodiment, a Cyanobacterial cell transformed with a nucleic acidconstruct is marker gene free. Methods for constructing recombinantmarker gene free microbial host cells are disclosed in EP-A-0 635 574and are based on the use of bidirectional markers. Alternatively, ascreenable marker such as Green Fluorescent Protein, lacZ, luciferase,chloramphenicol acetyltransferase, beta-glucuronidase may beincorporated into a nucleic acid construct of the invention allowing toscreen for transformed cells.

Optional further elements that may be present in a nucleic acidconstruct of the invention include, but are not limited to, one or moreleader sequences, enhancers, integration factors, and/or reporter genes,intron sequences, centromers, telomers and/or matrix attachment (MAR)sequences. A nucleic acid construct of the invention can be provided ina manner known per se, which generally involves techniques such asrestricting and linking nucleic acids/nucleic acid sequences, for whichreference is made to the standard handbooks, such as Sambrook and Russel(2001) “Molecular Cloning: A Laboratory Manual (3^(rd) edition), ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press.

Methods for inactivation and gene disruption in Cyanobacterial cells arewell known in the art (see e.g. Shestakov S V et al, (2002),Photosynthesis Research, 73: 279-284 and Nakamura Y et al, (1999),Nucleic Acids Res. 27:66-68).

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition the verb “to consist” may be replaced by“to consist essentially of” meaning that a peptide or a composition asdefined herein may comprise additional component(s) than the onesspecifically identified, said additional component(s) not altering theunique characteristic of the invention. In addition, reference to anelement by the indefinite article “a” or “an” does not exclude thepossibility that more than one of the element is present, unless thecontext clearly requires that there be one and only one of the elements.The indefinite article “a” or “an” thus usually means “at least one”.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety. The followingexamples are offered for illustrative purposes only, and are notintended to limit the scope of the present invention in any way

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: The light reaction reproduced from Berg, Tymoczko and Stryer:“Biochemistry” WH Freeman and Co, New York, 2006.

FIG. 2: The Calvin Cycle reproduced from Berg, Tymoczko and Stryer:“Biochemistry” WH Freeman and Co, New York, 1 2006.

FIG. 3: Construction of a recombinant strain: The cassette is introducedby homologous recombination and positioned downstream the SigEcontrolled promotor of Synechocystis PCC 6803. The black circle showsthe suicide plasmid (e.g. pBluescript) that is not able to replicate inSynechocystis PCC 6803. The cassette(s) of interest (denoted as arrow“x” in the figure) will be engineered to be flanked by DNA sequenceshomologous to a non-coding DNA region (shown as a dotted bar in thefigure) in the nrt operon. Via a double crossover event, shown in thefigure as dashed crosses, the cassettes of interest are integrated intothe Synechocystis genome. Alternatively, the construct can be insertedat a neutral docking site.

FIG. 4: Ammonium controlled production via the NtcA transcriptionalregulator. Conditions allowing growth repress production, ammoniumdepletion promotes production.

FIG. 5: Alcohol resistance. After five days, 100 μl of each culture wasdiluted and transferred to solid medium prepared from the BG-11 medium.Solid cultures were grown in an incubator at 30° C. under continuousillumination (70 μEinstein/m²/s) using two TL tubes without any addition(control) or with butanol, respectively ethanol added at variousconcentrations. After a week, single colonies were observed and counted.The amount of colonies was compared to the control sample.

FIG. 6: A preferred design of expression cassettes is given in FIG. 6.

EXAMPLES Example 1: Biochemical Background of the Cyanobacteria of theInvention

The energy, in the form of ATP, as well as the reductive power in theform of NADPH, that are both needed to drive the subsequent highlyendergonic dark reactions of photosynthesis, are catalyzed by the twophotosystems of oxygenic photosynthesis, PS-II and PS-I, arrangedaccording to the well-known Z-scheme, plus the membrane-bound ATPase(FIG. 1). In phototrophic organisms like Cyanobacteria, CO₂ is fixed inthe so-called Calvin cycle. This is a cyclic series of reductive stepsthat result in the net conversion of CO₂ into C3 compounds, such asglyceraldehyde-3-phosphate, phosphoglycerate and pyruvate (FIG. 2). Thispathway is essentially endergonic and in Nature driven by sunlight. Itconsumes CO₂ and water and yields C₃ compounds (e.g. pyruvate) andoxygen:

CO₂+H₂O+Solar energy→C₃ compounds+O₂  (1)

This reaction cannot proceed spontaneously: It is driven by theconsumption of the ATP and NADPH, generated in the ‘light’ reactions ofphotosynthesis. Subsequently, the C₃ compounds are used in Nature (i.e.in phototrophic organisms like plants and Cyanobacteria) as the buildingblocks to make new cells and/or plants. This requires additional amountsof reducing power (as NADPH) and energy conserved during the lightcapturing reactions (as ATP) and also allows the organisms toproliferate (grow and survive).

Nature also sustains an entirely different mode of (microbial) life:Numerous bacterial and fungal species are able to conserve sufficientenergy (as ATP) to proliferate by fermentation, in which they useso-called substrate level phosphorylation to generate their energy. Thisrespiration-independent mode of energy conservation relies on metabolicpathways that result in redox neutral dissimilation of the energysource. The most abundant pathways have evolved with sugars (e.g.glucose) as energy source and therefore all have glycolysis in common:

Glucose→glyceraldehyde-P→pyruvate+reducing power  (2)

Redox neutrality is maintained by the generalized reaction:

pyruvate+reducing power→fermentation products  (3)

Thus, it will contain the functional biochemistry to reduce theabove-mentioned intermediates to the end product and will have as itsbiocatalytic input and output the combination of (1) and (3),respectively:

CO₂+H₂O+Solar energy→organic product+O₂

Example 2: Description of the Expression System Used Genetic Cassettes

The identity of an organic product formed (and excreted into theenvironment) in the process of the invention depends on thespecies-specific gene cassettes (i.e. nucleic acid molecules representedby nucleotide sequences) that encode the respective biochemical pathway(see table 1). Preferred enzymes encoded by nucleic acid molecules aresubstantially oxygen insensitive.

TABLE 1 Examples of preferred donor organisms for the nucleic acidmolecules or genes to be introduced into a Cyanobacterium with thepathway they catalyze. For e.g. the production of ethanol andpropanediol various alternative donor organisms can be suggested. donorgenes pathway Sarcina ventriculi pyruvate decarboxylase, Pyruvate →acetaldehyde Lactobacillus brevis alcohol dehydrogenase Acetaldehyde →ethanol Clostridium thiolase pyruvate → acetoacetylCoA acetobutilicumhydroxybutyrylCoA dehydrogenase crotonase acetoacetylCoA → butyrylCoAbutyryl-CoA dehydrogenase butyrylCoA → butyraldehyde Butanoldehydrogenase butyraldehyde → 2-butanol Pseudomonas syringiae ethyleneforming enzyme 2-ketoglutarate → ethylene Lactococcus lactis lactatedehydrogenase pyruvate → D-lactate Lactococcus lactis acetolactatesynthase pyruvate → 2-acetolactate acetolactate decarboxylase2-acetolactate → acetoin diacetyl reductase diacetyl → acetoin acetoinreductase acetoin → 2,3 butanediol Klebsiella pneumoniae glyceroldehydratase 2,3 butanediol → 2-butanone 1,3 propanediol dehydrogenase2-butanone → 2-butanol Clostridium thiolase acetylCoA → acetoacetylCoAacetobutilicum ac.acetylCoA transferase acetoacetylCoA → acetoacetateacetoacetate decarboxylase acetoacetate → acetone Clostridium thiolaseacetylCoA → acetoacetylCoA acetobutilicum ac.acetylCoA transferaseacetoacetylCoA → acetoacetate acetoacetate decarboxylase acetoacetate →acetone Klebsiella pneumoniae propanol dehydrogenase acetone → propanolSynechocystis PCC glycerol-3-P dehydrogenase GAP → glycerol-P 6083glycerol-3-P Phosphatase glycerol-P → glycerol K. pneumoniae glyceroldehydratase glycerol → OHpropionaldehyde oxidoreductase OHprop.aldehyde→ 1,3-propanediol

Homologous Integration and Ammonium Controlled Expression

The genes/cassettes, necessary for the different pathways and respectiveorganic products in Synechocystis, are preferably introduced toSynechocystis via chromosomal integration. This will be achieved byhomologous recombination which allows to precisely define thechromosomal site of insertion. Appropriate plasmids for this purposeknown to be applicable in Synechocystis sp PCC 6830 are pBluescript(Stratagene, USA) or pGEM-T (Promega, USA). A strategy with respect tothe construct is exemplified in FIG. 3 but alternative (neutral) dockingsites for integration will be considered.

We will make use of the fact that expression of a number of glycolyticgenes of Synechocystis are under control of a group 2 sigma-factor,σ^(E). In turn, expression of the gene encoding this factor, SigE, isswitched on by the transcriptional regulator NtcA^(1,3). This switch is,amongst other unidentified signals, dependent on the extracellularnitrogen availability via the intracellular a-oxoglutarate/glutamateratio: under conditions of ammonium depletion of the medium to less than1 mM², NtcA binds to a-oxoglutarate and the resultingNtcA-a-oxoglutarate complex has a high binding affinity for and positivecontrol on the SigE promotor. Thus, a gene cassette under SigE controlwill be expressed upon ammonium depletion. As a consequence, duringammonium excess conditions, the carbon flux will be directed towardsbiosynthesis whereas in the stationary phase this flux will be directedto production (see FIG. 4). For Synechococcus the external ammoniumtreshold for the switch to σ^(E) synthesis was found to be submillimolarrange².

Example 3: Alcohol Resistance

Synechocystis PCC 6803 strain was grown on BG-11 medium (Stanier R Y, etal. Purification and properties of unicellular blue-green algae (orderChroococcales). Bacteriol. Rev. (1971) 35:171-205) in an orbital shakerat 30° C. under continuous illumination using two TL tubes, whichprovided average light intensity of approximately 70 μE·m⁻²·s⁻¹. Toquantify the influence of alcohols on the net growth rate, cells weregrown without any addition (control) or with butanol, respectivelyethanol added at various concentrations.

After 5 days, 100 μl of each culture was appropriately diluted andtransferred to solid medium prepared from BG-11 and supplemented with0.3% sodium thiosulfate, 10 mMN-tris[hydroxymethyl]-2-aminoethanesulfonic acid (TES) pH 8.2, 5 mMglucose and 1.5% bactoagar. Solid cultures were grown in an incubator30° C. under continuous illumination. After a week, single colonies wereobserved and counted. The amount of colonies was compared to the controlsample.

From the results shown below in FIG. 5, it is concluded that the netspecific growth rate decreases linearly with increasing alcoholconcentration and that the growth rate is reduced by 50% atconcentrations of approximately 0.17 M butanol respectively 0.29 Methanol. Therefore, it is to be expected that the two phases productionprocess of the invention is much more efficient than a single phaseproduction process.

TABLE 3 list of all primers used HOMOLOGY REGION 1 SEQ ID NO: ForwardAAATGGTACCGAACTGAGATTAGCCCCGGAC KpnI  37 ReverseAAATCTCGAGACCAGGACATCCGACTTGC XhoI  38 HOMOLOGY REGION 2 ForwardCACGACTAGTGTGACCGGGTCATTTTTTTGCTATTTATTCC SpeI  39 ReverseAAATTCTAGATAACTGCGGTAGCACTAAAGCCGCTGCCTTAG XbaI  40 Product: Lactic acidForward 1dh CAATCTCGAGATGGCTGATAAACAACGTAAG XhoI  41 Reverse 1dhCAATGAATTCTTAGTTTTTAACTGCAGAAGCAAATTC EcoRI  42 Product: EthanolForward pdc ATAACTCGAGGACAATAGGTGCTTTAATCAC XhoI  43 Reverse pdcCGACGATATCAGGTGTAAAATACCATTTATTAAAATAG EcoRV  44 Forward adhCATTGATATCATGTCTAACCGTTTGGATGG EcoRV  45 Reverse adhCATACTGCAGCTATTGAGCAGTGTAGCCAC PstI  46 Product: 1,3-PropanediolForward gpd AAATCTCGAGTCAGTGGAGACAATAGTCG XhoI  47 Reverse gpdAAATATCGATATGCGTAATTTCCCAGAAATC ClaI  48 Forward dhakCATAAAGCTTATGAAATTCTATACTTCAACGACAG HindIII  49 Reverse dhakAAATGATATCTTACCAGGCGAAAGCTC EcoRV  50 Forward G1 dehydrAAATATCGATTTATTCAATGGTGTCAGGCTG ClaI  51 Reverse G1 dehydrCCAAAAGCTTATGAAAAGATCAAAACGATTTG HindIII  52 ForwardGGGTGATATCTTAAGGTAAAGTAAAGTCAACCCAC EcoRV  53 oxidoreductase ReverseAAATGAATTCATGTTAAACGGCCTGAAAC EcoRI  54 oxidoreductaseLactic acid-II set of primers SEQ ID NO: ForwardAAATGGTACCGAACTGAGATTAGCCCCGGAC KpnI  55 HomologyI ReverseGTTGTTTATCAGCCATACCAGGACATCCGACTTG  56 HomologyI Reverse forCTGCGTGCAATCCATCTTGTTCAATCATTTAGTTTTTAACTGCAGAAGCAAATTC  57 ldhReverse for GCAAAAAAATGACCCGGTCACTCAGAAGAACTCGTCAAGAAGG  58 KANReverse for AAATTCTAGATAACTGCGGTAGCACTAAAGCCGCTGCCTTAC XbaI  59HomologyII Ethanol-II set of primers ForwardAAATGGTACCGAACTGAGATTAGCCCCGGAC KpnI  60 HomologyI ReverseGATTAAAGCACCTATTGTCACCAGGACATCCGACTTG  61 HomologyII Reverse pdcCTACCTTACCATCCAAACGGTTAGACATAGGTGTAAAATACCATTTATTAAAATAG  62 Reverse adhCAATCCATCTTGTTCAATCATCTATTGAGCAGTGTAGCCACCGTC  63 Reverse KANGCAAAAAAATGACCCGGTCACTCAGAAGAACTCGTCAAGAAGG  64 ReverseAAATTCTAGATAACTGCGGTAGCACTAAAGCCGCTGCCTTAC XbaI  65 HomologyII1,3-Propanediol Forward AAATGGTACCGAACTGAGATTAGCCCCGGAC KpnI  66HomologyI Reverse TATTGTCTCCACTGAACCAGGACATCCGACTTG  67 HomologyIReverse dhg CTGTCGTTGAAGTATAGAATTTCATATGCGTAATTTCCCAGAAATCCAAAATACG  68Reverse dha k GGTTCAGCCTGACACCATTGAATAATTACCAGGCGAAAGCTCCAGTTGGAGC  69Reverse GTGGTTGACTTTACTTTACCTTAAATGAAAAGATCAAAACGATTTGCAGTACTGG  70glycerol dehydrts Reverse CAATCCATCTTGTTCAATCATATGTTAAACGGCCTGAAACC  71oxidored Reverse KAN GCAAAAAAATGACCCGGTCACTCAGAAGAACTCGTCAAGAAGG  72Reverse AAATTCTAGATAACTGCGGTAGCACTAAAGCCGCTGCCTTAC XbaI  73 HomologyIIEthylene Forward Efe TAAAGTCGACAAGGAGACTAGCATGACCAAC SalI 135Reverse Efe TAAAGAATTCTTAGGAGCCGGTGG EcoRI  94 2-Butanol (Clostridium)forward thl AAGGAGATTCCAATGAGAGATGTAGTAATAGTAAG  99 reverse thlTTAGTCTCTTTCAACTACGAGAGCTGTTCCCTG 100 forward 3bdhAAGGAGATTCCAATGAAAAAGGTATGTGTTATAG 101 reverse 3bdhTTATTTTGAATAATCGTAGAAACCTTTTCCTG 102 forward crt-etfAAGGAGATTCCAATGTCAAAAGAGATTTATGAATCAG 103 reverse crt-etfCTACAATTTTTTTACCAAATTCAAAAACATTCC 104 forward aldAAGGAGATTCCAATGGATTTTAATTTAACAAGAG 105 reverse aldTTATCTAAAAATTTTCCTGAAATAACTAATTTTCTGAACTTC 106 forward bdhAAGGAGATTCCAATGCTAAGTTTTGATTATTCAATAC 107 reverse bdhTTAATATGATTTTTTAAATATCTCAAGAAGCATCCTCTG 1082-Butanol (L. lactis and K. pneumoniae) Foreward L.AAGGAGACTACTATGTCTGAGAAACAATTTGGGGC 109 lactis als Reverse L.TCAGTAAAATTCTTCTGGCAAT 110 lactis als Foreward L.AAGGAGACTACTATGTCAGAAATCACACAACTTTTTCA 111 lactis aldB Reverse L.TCATTCAGCTACATCAATATCTTTTTTCAAAGC 112 lactis aldB Foreward L.AAGGAGACTACTATGTCTAAAGTTGCAGCAGTTACTGG 113 lactis butA Reverse L.TTAATGAAATTGCATTCCACCATC 114 lactis butA Foreward L.AAGGAGACTACTGTGGCTTGGTGTGGAATCTGT 115 lactis butB Reverse L.TTATAGACCTTTTCCAGTTGGTG 116 lactis butB Foreward K.AAGGAGACTACTATGAAAAGATCAAAACGATTTGCAG 117 pneumoniae dhaB Reverse K.TCAGAATGCCTGGCGGAAAAT 118 pneumoniae dhaB Foreward K.AAGGAGACTACTATGAGCTATCGTATGTTTGATTATCTGG 119 pneumoniae dhaT Reverse K.TCAGAATGCCTGGCGGAAAAT 120 pneumoniae dhaT Acetone Foreward thlAAGGAGATTCCAATGAGAGATGTAGTAATAGTAAG 121 Reverse thlTTAGTCTCTTTCAACTACGAGAGCTGTTCCCTG 122 Foreward ctfABAAGGAGGCGGCGATGAACTCTAAAATAATTAG 123 Reverse ctfABTTATGCAGGCTCCTTTACTATATAAT 124 Foreward adcAAGGAGGCGGCGATGTTAAAGGATGAAGTA 125 Reverse adcCCCTTACTTAAGATAATCATATATAACTTCAGC 126 Propanol Foreward thlAAGGAGATTCCAATGAGAGATGTAGTAATAGTAAG 127 Reverse thlTTAGTCTCTTTCAACTACGAGAGCTGTTCCCTG 128 Foreward ctfABAAGGAGGCGGCGATGAACTCTAAAATAATTAG 129 Reverse ctfABTTATGCAGGCTCCTTTACTATATAAT 130 Foreward adcAAGGAGGCGGCGATGTTAAAGGATGAAGTA 131 Reverse adcCCCTTACTTAAGATAATCATATATAACTTCAGC 132 Foreward K.AAGGAGAATTCCAATGCATACCTTTTCTCTGC 133 pneumoniae aad Reverse K.TCATTGCAGGTTCTCCAGCAGTTGC 134 pneumoniae aad

REFERENCES

-   ¹Aichi, M., Takatani N., Omata T. (2001) Role of NtcB in activation    of Nitrate ssimilation Genes in the Cyanobacterium Synechocystis sp.    Strain PCC6803. J. Bacteriol. 183, 5840-5847-   ²Gillor, O., Harush, A., Post, A. F., Belkin, S. (2003) A    Synechococcus PglnA::luxAB fusion for estimation of nitrogen    bioavailability to freshwater cyanobacteria. Appl. Environm.    Microbiol. 69, 1465-1474-   ³Osanai, T., Imamura, S., Asayama, M., Shirai, M., Suzuki, I.,    Murata, N., Tanaka, K, (2006) Nitrogen induction of sugar catabolic    gene expression in Synechocystis sp. PCC 6803. DNA Research 13,    185-195

1. A process of producing an organic compound and/or an intermediarycompound produced in a pathway leading to said organic compound,comprising feeding carbon dioxide to a culture of a Cyanobacteria celland subjecting said culture to light, wherein said cell is capable ofexpressing a nucleic acid molecule, wherein the expression of saidnucleic acid molecule confers on the cell an ability to convert aglycolytic intermediate into said organic compound and/or into saidintermediary compound, and wherein said nucleic acid molecule is underthe control of a regulatory system which responds to a change in theconcentration of a nutrient in said culture.
 2. The process according toclaim 1, wherein said enzyme is substantially not sensitive towardsoxygen inactivation.
 3. The process according to claim 1, wherein theorganic product is butanol and the intermediary compound isbutyraldehyde, and wherein the nucleic acid molecule codes for at leastone enzyme capable of converting pyruvate to butanol selected from thegroup consisting of: a thiolase, a hydroxybutyrylCoA dehydrogenase, acrotonase, a butyrylCoA dehdyrogenase, a butyraldehyde dehydrogenase,and a butanol dehdyrogenase.
 4. The process according to claim 1,wherein the organic product is butanol and the intermediary compound isproduced in the pathway leading to butanol, and wherein the nucleic acidmolecule codes for at least one enzyme capable of converting pyruvate tobutanol selected from the group consisting of: a 2-acetolactatesynthetase, an acetolactate decarboxylase, a diacetyl reductase, anacetoin reductase, a glycerol dehydratase, and a 1,3-propanedioldehydrogenase.
 5. The process according to claim 1, wherein the organicproduct is ethanol and the intermediary compound is acetaldehyde, andwherein the nucleic acid molecule codes for at least one enzyme capableof converting pyruvate into ethanol selected from the group consistingof a pdc and an alcohol dehydrogenase.
 6. The process according to claim1, wherein the organic product is propanol and the intermediary compoundis acetone, and wherein the nucleic acid molecule codes for at least oneenzyme capable of converting pyruvate to propanol selected from thegroup consisting of: a thiolase, an acetoacetylCoA transferase, anacetoacetylCoA decarboxylase, and a propanol dehydrogenase.
 7. Theprocess according to claim 1, wherein the organic product is acetone,and wherein the nucleic acid molecule codes for at least one enzymecapable of converting pyruvate to acetone selected from the groupconsisting of: a thiolase, an acetoacetylCoA transferase, and anacetoacetylCoA decarboxylase.
 8. The process according to claim 1,wherein the organic product is 1,3-propanediol and the intermediarycompound is glycerol and/or hydroxypropionaldehyde, and wherein thenucleic acid molecule codes for at least one enzyme capable ofconverting glyceraldehyde-3-phosphate to propanediol selected from thegroup consisting of: a glycerol-3-P-dehydrogenase, a glycerol-3-Pphosphatase, a glycerol dehydratase, and an oxidoreductase.
 9. Theprocess according to claim 1, wherein the organic product is D-lactate,and wherein the nucleic acid molecule codes for at least one enzymecapable of converting pyruvate to D-lactate and comprises a lactatedehydrogenase.
 10. The process according to claim 1, wherein the organicproduct is ethylene, and wherein the nucleic acid molecule code for atleast one enzyme capable of converting 2-oxoglutarate into ethylene andsuccinate.
 11. The process according to claim 1, wherein the regulatorysystem responds to a change in concentration of a nutrient ammonium insaid culture, and/or wherein said nucleic acid molecule is integratedinto the Cyanobacteria genome, preferably via homologous recombination,and/or wherein the Cyanobacteria cell is derived from a Synechocystiscell, preferably a Synechocystis PCC 6803 cell.
 12. The processaccording to claim 1, wherein the organic product and/or intermediarycompound is separated from the culture.
 13. A Cyanobacteria capable ofexpressing a nucleic acid molecule, wherein the expression of saidnucleic acid molecule confers on the Cyanobacteria the ability toconvert a glycolytic intermediate into an organic compound and/or intoan intermediary compound produced in a pathway leading to the organicproduct, and wherein the nucleic acid molecule is under the control of aregulatory system which responds to a change in the concentration of anutrient when culturing said Cyanobacteria.
 14. The Cyanobacteriaaccording to claim 13, wherein the glycolytic intermediate isglyceraldehyde-3-phosphate or pyruvate, the organic compound is selectedfrom the group consisting of butanol, ethanol, propanol,1,3-propanediol, acetone, D-lactate and ethylene, the intermediaryproduct is selected from the group consisting of butyraldehydeacetaldehyde, acetone, glycerol and hydroxypropionaldehyde, and theenzyme is selected from the group consisting of a thiolase, ahydroxybutyrylCoA dehydrogenase, a crotonase, a butyrylCoAdehdyrogenase, a butyraldehyde dehydrogenase, a butanoldehdyrogenase2-acetolactate synthetase, an acetolactate decarboxylase, adiacetyl reductase, an acetoin reductase, a glycerol dehydratase, a1,3-propanediol dehydrogenase, a pdc, an alcohol dehydrogenase, anacetoacetylCoA transferase, an acetoacetylCoA decarboxylase, a propanoldehydrogenase, a glycerol-3-P-dehydrogenase, a glycerol-3-P phosphatase,a glycerol dehydratase, an oxidoreductase, and a lactate dehydrogenase.15. The Cyanobacteria according to claim 13, wherein the regulatorysystem responds to a change in concentration of a nutrient ammonium,and/or wherein said nucleic acid molecule is integrated into theCyanobacteria genome, preferably via homologous recombination, and/orwherein the Cyanobacteria is derived from Synechocystis, preferablySynechocystis PCC 6803.